Modulation of the rate, enantioselectivity, and substrate specificity of semisynthetic transaminases based on lipid binding proteins using site directed mutagenesis

Fatty acid binding proteins are a class of small 15 kDa proteins with a simple architecture that forms a large solvent sequestered cavity. In previous work, we demonstrated that reductive amination reactions could be performed in this cavity by covalent attachment of a pyridoxamine cofactor to the protein. Here, we report the results of experiments in which the position of pyridoxamine attachment has been varied by site directed mutagenesis. The conjugate IFABP-PX60 reacts at least 9.4-fold more rapidly than our original conjugate ALBP-PX, while IFABP-PX72 inverts the enantioselectivity of reactions (compared to ALBP-PX) and IFABP-PX104 displays very selective substrate specificty. These results indicate that site-directed mutagenesis can be used to tune the rate, enantioselectivity, and substrate specificity of semisynthetic transaminases based on fatty acid binding proteins.